Ros0300

To measure the effect of the ADBR2-blockade on ROS production in 786-O and ccRCC primary cultures, the DCF-DA solution reagent was used (ROS0300, OzBiosciences, San Diego, CA, USA).
Briefly, 5 × 10³ cells/well were seeded in a 96-well plate and incubated in complete medium with [0–50 μM for 786-O and 0–100 μM for ccRCC] propranolol or ICI. After 72 h, cells were washed with PBS and then incubated with 100 μL DCF-DA for 30 min at 37 °C in darkness. After DCF-DA removal, 100 μL/well PBS was added and fluorescence (exc: 485 nm/em: 535 nm) was measured using a GLOMAX multi-detection system (Promega). Fluorescence signal was normalized to cell number.
Bright field and fluorescence microscopy images from the same samples were taken using a Pantera microscope and the Motic Images Plus 3.0 software (Motic, Wetzlar, Germany). FIJI-Image J software tool (NIH, Bethesda, MD, USA) was used to process and quantify the fluorescence intensities.

To measure reactive oxygen species (ROS) production, 5 × 10³ BOECs (control and VHL) per well were seeded in a 96-well plate. After 72 h, cells were washed with PBS twice and stained by adding 100 μL of diluted DCF-DA solution per well. After 30 min of incubation at 37 °C in the dark, DCF-DA was removed and cells washed. Then, 100 μL/well PBS was added and fluorescence measured (exc: 485 nm/em: 535 nm) (ROS0300, OzBiosciences, San Diego, CA, USA).